A minimal presynaptic protein machinery mediating synchronous and asynchronous exocytosis and short-term plasticity.

Neurotransmitters are released from synaptic vesicles with remarkable precision in response to presynaptic Ca2+ influx but exhibit significant heterogeneity in exocytosis timing and efficacy based on the recent history of activity. This heterogeneity is critical for information transfer in the brain, yet its molecular basis remains poorly understood. Here, we employ a biochemically-defined fusion assay under physiologically-relevant conditions to delineate the minimal protein machinery sufficient to account for different modes of Ca2+-triggered vesicle fusion and short-term facilitation. We find that Synaptotagmin-1, Synaptotagmin-7, and Complexin, synergistically restrain SNARE complex assembly, thus preserving vesicles in a stably docked state at rest. Upon Ca2+ activation, Synaptotagmin-1 induces rapid vesicle fusion, while Synaptotagmin-7 mediates delayed fusion. Competitive binding of Synaptotagmin-1 and Synaptotagmin-7 to the same SNAREs, coupled with differential rates of Ca2+-triggered fusion clamp reversal, govern the kinetics of vesicular fusion. Under conditions mimicking sustained neuronal activity, the Synaptotagmin-7 fusion clamp is destabilized by the elevated basal Ca2+ concentration, thereby enhancing the synchronous component of fusion. These findings provide a direct demonstration that a small set of proteins is sufficient to account for how nerve terminals adapt and regulate the Ca2+-evoked neurotransmitter exocytosis process to support their specialized functions in the nervous system.

Synaptophysin chaperones the assembly of 12 SNAREpins under each ready-release vesicle.

The synaptic vesicle protein Synaptophysin (Syp) has long been known to form a complex with the Vesicle associated soluble N-ethylmaleimide sensitive fusion protein attachment receptor (v-SNARE) Vesicle associated membrane protein (VAMP), but a more specific molecular function or mechanism of action in exocytosis has been lacking because gene knockouts have minimal effects. Utilizing fully defined reconstitution and single-molecule measurements, we now report that Syp functions as a chaperone that determines the number of SNAREpins assembling between a ready-release vesicle and its target membrane bilayer. Specifically, Syp directs the assembly of 12 ± 1 SNAREpins under each docked vesicle, even in the face of an excess of SNARE proteins. The SNAREpins assemble in successive waves of 6 ± 1 and 5 ± 2 SNAREpins, respectively, tightly linked to oligomerization of and binding to the vesicle Ca++ sensor Synaptotagmin. Templating of 12 SNAREpins by Syp is likely the direct result of its hexamer structure and its binding of VAMP2 dimers, both of which we demonstrate in detergent extracts and lipid bilayers.

Roles for diacylglycerol in synaptic vesicle priming and release revealed by complete reconstitution of core protein machinery.

Here, we introduce the full functional reconstitution of genetically validated core protein machinery (SNAREs, Munc13, Munc18, Synaptotagmin, and Complexin) for synaptic vesicle priming and release in a geometry that enables detailed characterization of the fate of docked vesicles both before and after release is triggered with Ca2+. Using this setup, we identify new roles for diacylglycerol (DAG) in regulating vesicle priming and Ca2+-triggered release involving the SNARE assembly chaperone Munc13. We find that low concentrations of DAG profoundly accelerate the rate of Ca2+-dependent release, and high concentrations reduce clamping and permit extensive spontaneous release. As expected, DAG also increases the number of docked, release-ready vesicles. Dynamic single-molecule imaging of Complexin binding to release-ready vesicles directly establishes that DAG accelerates the rate of SNAREpin assembly mediated by chaperones, Munc13 and Munc18. The selective effects of physiologically validated mutations confirmed that the Munc18-Syntaxin-VAMP2 "template" complex is a functional intermediate in the production of primed, release-ready vesicles, which requires the coordinated action of Munc13 and Munc18.

Turbocharging synaptic transmission.

Evidence from biochemistry, genetics, and electron microscopy strongly supports the idea that a ring of Synaptotagmin is central to the clamping and release of synaptic vesicles (SVs) for synchronous neurotransmission. Recent direct measurements in cell-free systems suggest there are 12 SNAREpins in each ready-release vesicle, consisting of six peripheral and six central SNAREpins. The six central SNAREpins are directly bound to the Synaptotagmin ring, are directly released by Ca++ , and they initially open the fusion pore. The six peripheral SNAREpins are indirectly bound to the ring, each linked to a central SNAREpin by a bridging molecule of Complexin. We suggest that the primary role of peripheral SNAREpins is to provide additional force to 'turbocharge' neurotransmitter release, explaining how it can occur much faster than other forms of membrane fusion. The SV protein Synaptophysin forms hexamers that bear two copies of the v-SNARE VAMP at each vertex, one likely assembling into a peripheral SNAREpin and the other into a central SNAREpin.

Two successive oligomeric Munc13 assemblies scaffold vesicle docking and SNARE assembly to support neurotransmitter release.

The critical presynaptic protein Munc13 serves numerous roles in the process of docking and priming synaptic vesicles. Here we investigate the functional significance of two distinct oligomers of the Munc13 core domain (Munc13C) comprising C1-C2B-MUN-C2C. Oligomer interface point mutations that specifically destabilized either the trimer or lateral hexamer assemblies of Munc13C disrupted vesicle docking, trans-SNARE formation, and Ca 2+ -triggered vesicle fusion in vitro and impaired neurotransmitter secretion and motor nervous system function in vivo. We suggest that a progression of oligomeric Munc13 complexes couples vesicle docking and assembly of a precise number of SNARE molecules to support rapid and high-fidelity vesicle priming.

Synaptophysin Chaperones the Assembly of 12 SNAREpins under each Ready-Release Vesicle.

The synaptic vesicle protein Synaptophysin has long been known to form a complex with the v-SNARE VAMP, but a more specific molecular function or mechanism of action in exocytosis has been lacking because gene knockouts have minimal effects. Utilizing fully-defined reconstitution and single-molecule measurements, we now report that Synaptophysin functions as a chaperone that determines the number of SNAREpins assembling between a ready-release vesicle and its target membrane bilayer. Specifically, Synaptophysin directs the assembly of 12 ± 1 SNAREpins under each docked vesicle, even in the face of an excess of SNARE proteins. The SNAREpins assemble in successive waves of 6 ± 1 and 5 ± 2 SNAREpins, respectively, tightly linked to oligomerization of and binding to the vesicle Ca++ sensor Synaptotagmin. Templating of 12 SNAREpins by Synaptophysin is likely the direct result of its hexamer structure and its binding of VAMP2 dimers, both of which we demonstrate in detergent extracts and lipid bilayers.

Rapid Quantification of First and Second Phase Insulin Secretion Dynamics using an In vitro Platform for Improving Insulin Therapy.

High-throughput quantification of the first- and second-phase insulin secretion dynamics is intractable with current methods. The fact that independent secretion phases play distinct roles in metabolism necessitates partitioning them separately and performing high-throughput compound screening to target them individually. We developed an insulin-nanoluc luciferase reporter system to dissect the molecular and cellular pathways involved in the separate phases of insulin secretion. We validated this method through genetic studies, including knockdown and overexpression, as well as small-molecule screening and their effects on insulin secretion. Furthermore, we demonstrated that the results of this method are well correlated with those of single-vesicle exocytosis experiments conducted on live cells, providing a quantitative reference for the approach. Thus, we have developed a robust methodology for screening small molecules and cellular pathways that target specific phases of insulin secretion, resulting in a better understanding of insulin secretion, which in turn will result in a more effective insulin therapy through the stimulation of endogenous glucose-stimulated insulin secretion.

Novel Roles for Diacylglycerol in Synaptic Vesicle Priming and Release Revealed by Complete Reconstitution of Core Protein Machinery.

Here we introduce the full functional reconstitution of genetically-validated core protein machinery (SNAREs, Munc13, Munc18, Synaptotagmin, Complexin) for synaptic vesicle priming and release in a geometry that enables detailed characterization of the fate of docked vesicles both before and after release is triggered with Ca 2+ . Using this novel setup, we discover new roles for diacylglycerol (DAG) in regulating vesicle priming and Ca 2+- triggered release involving the SNARE assembly chaperone Munc13. We find that low concentrations of DAG profoundly accelerate the rate of Ca 2+ -dependent release, and high concentrations reduce clamping and permit extensive spontaneous release. As expected, DAG also increases the number of ready-release vesicles. Dynamic single-molecule imaging of Complexin binding to ready-release vesicles directly establishes that DAG accelerates the rate of SNAREpin assembly mediated by Munc13 and Munc18 chaperones. The selective effects of physiologically validated mutations confirmed that the Munc18-Syntaxin-VAMP2 'template' complex is a functional intermediate in the production of primed, ready-release vesicles, which requires the coordinated action of Munc13 and Munc18. SIGNIFICANCE STATEMENT: Munc13 and Munc18 are SNARE-associated chaperones that act as "priming" factors, facilitating the formation of a pool of docked, release-ready vesicles and regulating Ca 2+ -evoked neurotransmitter release. Although important insights into Munc18/Munc13 function have been gained, how they assemble and operate together remains enigmatic. To address this, we developed a novel biochemically-defined fusion assay which enabled us to investigate the cooperative action of Munc13 and Munc18 in molecular terms. We find that Munc18 nucleates the SNARE complex, while Munc13 promotes and accelerates the SNARE assembly in a DAG-dependent manner. The concerted action of Munc13 and Munc18 stages the SNARE assembly process to ensure efficient 'clamping' and formation of stably docked vesicles, which can be triggered to fuse rapidly (∼10 msec) upon Ca 2+ influx.

Native Planar Asymmetric Suspended Membrane for Single-Molecule Investigations: Plasma Membrane on a Chip.

Cellular plasma membranes, in their role as gatekeepers to the external environment, host numerous protein assemblies and lipid domains that manage the movement of molecules into and out of cells, regulate electric potential, and direct cell signaling. The ability to investigate these roles on the bilayer at a single-molecule level in a controlled, in vitro environment while preserving lipid and protein architectures will provide deeper insights into how the plasma membrane works. A tunable silicon microarray platform that supports stable, planar, and asymmetric suspended lipid membranes (SLIM) using synthetic and native plasma membrane vesicles for single-molecule fluorescence investigations is developed. Essentially, a "plasma membrane-on-a-chip" system that preserves lipid asymmetry and protein orientation is created. By harnessing the combined potential of this platform with total internal reflection fluorescence (TIRF) microscopy, the authors are able to visualize protein complexes with single-molecule precision. This technology has widespread applications in biological processes that happen at the cellular membranes and will further the knowledge of lipid and protein assemblies.

Molecular determinants of complexin clamping and activation function.

Previously we reported that Synaptotagmin-1 and Complexin synergistically clamp the SNARE assembly process to generate and maintain a pool of docked vesicles that fuse rapidly and synchronously upon Ca2+ influx (Ramakrishnan et al., 2020). Here, using the same in vitro single-vesicle fusion assay, we determine the molecular details of the Complexin-mediated fusion clamp and its role in Ca2+-activation. We find that a delay in fusion kinetics, likely imparted by Synaptotagmin-1, is needed for Complexin to block fusion. Systematic truncation/mutational analyses reveal that continuous alpha-helical accessory-central domains of Complexin are essential for its inhibitory function and specific interaction of the accessory helix with the SNAREpins enhances this functionality. The C-terminal domain promotes clamping by locally elevating Complexin concentration through interactions with the membrane. Independent of their clamping functions, the accessory-central helical domains of Complexin also contribute to rapid Ca2+-synchronized vesicle release by increasing the probability of fusion from the clamped state.

RETREG1/FAM134B mediated autophagosomal degradation of AMFR/GP78 and OPA1 -a dual organellar turnover mechanism.

Turnover of cellular organelles, including endoplasmic reticulum (ER) and mitochondria, is orchestrated by an efficient cellular surveillance system. We have identified a mechanism for dual regulation of ER and mitochondria under stress. It is known that AMFR, an ER E3 ligase and ER-associated degradation (ERAD) regulator, degrades outer mitochondrial membrane (OMM) proteins, MFNs (mitofusins), via the proteasome and triggers mitophagy. We show that destabilized mitochondria are almost devoid of the OMM and generate "mitoplasts". This brings the inner mitochondrial membrane (IMM) in the proximity of the ER. When AMFR levels are high and the mitochondria are stressed, the reticulophagy regulatory protein RETREG1 participates in the formation of the mitophagophore by interacting with OPA1. Interestingly, OPA1 and other IMM proteins exhibit similar RETREG1-dependent autophagosomal degradation as AMFR, unlike most of the OMM proteins. The "mitoplasts" generated are degraded by reticulo-mito-phagy - simultaneously affecting dual organelle turnover.Abbreviations: AMFR/GP78: autocrine motility factor receptor; BAPTA: 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; BFP: blue fluorescent protein; CCCP: carbonyl cyanide m-chlorophenyl hydrazone; CNBr: cyanogen bromide; ER: endoplasmic reticulum; ERAD: endoplasmic-reticulum-associated protein degradation; FL: fluorescence, GFP: green fluorescent protein; HA: hemagglutinin; HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; IMM: inner mitochondrial membrane; LIR: LC3-interacting region; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MFN: mitofusin, MGRN1: mahogunin ring finger 1; NA: numerical aperature; OMM: outer mitochondrial membrane; OPA1: OPA1 mitochondrial dynamin like GTPase; PRNP/PrP: prion protein; RER: rough endoplasmic reticulum; RETREG1/FAM134B: reticulophagy regulator 1; RFP: red fluorescent protein; RING: really interesting new gene; ROI: region of interest; RTN: reticulon; SEM: standard error of the mean; SER: smooth endoplasmic reticulum; SIM: structured illumination microscopy; SQSTM1/p62: sequestosome 1; STED: stimulated emission depletion; STOML2: stomatin like 2; TOMM20: translocase of outer mitochondrial membrane 20; UPR: unfolded protein response.

Pulling the springs of a cell by single-molecule force spectroscopy.

The fundamental unit of the human body comprises of the cells which remain embedded in a fibrillar network of extracellular matrix proteins which in turn provides necessary anchorage the cells. Tissue repair, regeneration and reprogramming predominantly involve a traction force mediated signalling originating in the ECM and travelling deep into the cell including the nucleus via circuitry of spring-like filamentous proteins like microfilaments or actin, intermediate filaments and microtubules to elicit a response in the form of mechanical movement as well as biochemical changes. The 'springiness' of these proteins is highlighted in their extension-contraction behaviour which is manifested as an effect of differential traction force. Atomic force microscope (AFM) provides the magic eye to visualize and quantify such force-extension/indentation events in these filamentous proteins as well as in whole cells. In this review, we have presented a summary of the current understanding and advancement of such measurements by AFM based single-molecule force spectroscopy in the context of cytoskeletal and nucleoskeletal proteins which act in tandem to facilitate mechanotransduction.

Synergistic roles of Synaptotagmin-1 and complexin in calcium-regulated neuronal exocytosis.

Calcium (Ca2+)-evoked release of neurotransmitters from synaptic vesicles requires mechanisms both to prevent un-initiated fusion of vesicles (clamping) and to trigger fusion following Ca2+-influx. The principal components involved in these processes are the vesicular fusion machinery (SNARE proteins) and the regulatory proteins, Synaptotagmin-1 and Complexin. Here, we use a reconstituted single-vesicle fusion assay under physiologically-relevant conditions to delineate a novel mechanism by which Synaptotagmin-1 and Complexin act synergistically to establish Ca2+-regulated fusion. We find that under each vesicle, Synaptotagmin-1 oligomers bind and clamp a limited number of 'central' SNARE complexes via the primary interface and introduce a kinetic delay in vesicle fusion mediated by the excess of free SNAREpins. This in turn enables Complexin to arrest the remaining free 'peripheral' SNAREpins to produce a stably clamped vesicle. Activation of the central SNAREpins associated with Synaptotagmin-1 by Ca2+ is sufficient to trigger rapid (<100 msec) and synchronous fusion of the docked vesicles.

Nuclear filaments: role in chromosomal positioning and gene expression.

Nuclear lamins form an elastic meshwork underlying the inner nuclear membrane and provide mechanical rigidity to the nucleus and maintain shape. Lamins also maintain chromosome positioning and play important roles in several nuclear processes like replication, DNA damage repair, transcription, and epigenetic modifications. LMNA mutations affect cardiac tissue, muscle tissues, adipose tissues to precipitate several diseases collectively termed as laminopathies. However, the rationale behind LMNA mutations and laminopathies continues to elude scientists. During interphase, several chromosomes form inter/intrachromosomal contacts inside nucleoplasm and several chromosomal loops also stretch out to make a 'loop-cluster' which are key players to regulate gene expressions. In this perspective, we have proposed that the lamin network in tandem with nuclear actin and myosin provide mechanical rigidity to the chromosomal contacts and facilitate loop-clusters movements. LMNA mutations thus might perturb the landscape of chromosomal contacts or loop-clusters positioning which can impair gene expression profile.

Chromosome Territorial Organization Drives Efficient Protein Complex Formation: A Hypothesis.

In eukaryotes, chromosomes often form a transcriptional kissing loop during interphase. We propose that these kissing loops facilitate the formation of protein complexes. mRNA transcripts from these loops could cluster together into phase-separated nuclear granules. Their export into the ER could be ensured by guided diffusion through the inter-chromatin space followed by association with nuclear baskets and export factors. Inside the ER, these mRNAs would form a translation hub. Juxtaposed translation of these mRNAs would increase the cis/trans protein complex assembly among the nascent protein chains. Eukaryotes might employ this pathway to increase complex formation efficiency.

Synaptotagmin oligomers are necessary and can be sufficient to form a Ca2+ -sensitive fusion clamp.

The buttressed-ring hypothesis, supported by recent cryo-electron tomography analysis of docked synaptic-like vesicles in neuroendocrine cells, postulates that prefusion SNAREpins are stabilized and organized by Synaptotagmin (Syt) ring-like oligomers. Here, we use a reconstituted single-vesicle fusion analysis to test the prediction that destabilizing the Syt1 oligomers destabilizes the clamp and results in spontaneous fusion in the absence of Ca2+ . Vesicles in which Syt oligomerization is compromised by a ring-destabilizing mutation dock and diffuse freely on the bilayer until they fuse spontaneously, similar to vesicles containing only v-SNAREs. In contrast, vesicles containing wild-type Syt are immobile as soon as they attach to the bilayer and remain frozen in place, up to at least 1 h until fusion is triggered by Ca2+ .

Significance of 1B and 2B domains in modulating elastic properties of lamin A.

Nuclear lamins are type V intermediate filament proteins which form an elastic meshwork underlying the inner nuclear membrane. Lamins directly contribute to maintain the nuclear shape and elasticity. More than 400 mutations have been reported in lamin A that are involved in diseases known as laminopathies. These mutations are scattered mainly in the lamin rod domain along with some in its C-terminal domain. The contribution of the rod domain towards the elasticity of lamin A molecule was hitherto unknown. Here, we have elucidated the significance of the 1B and 2B domains of the rod in modulating the elastic behavior of lamin A by single-molecule force spectroscopy. In addition, we have also studied the network forming capacity of these domains and their corresponding viscoelastic behavior. We have shown that the 1B domain has the ability to form a lamin-like network and resists larger deformation. However at the single-molecular level, both the domains have comparable mechanical properties. The self-assembly of the 1B domain contributes to the elasticity of the lamin A network.

Characterization of unfolding mechanism of human lamin A Ig fold by single-molecule force spectroscopy-implications in EDMD.

A- and B-type lamins are intermediate filament proteins constituting the nuclear lamina underneath the nuclear envelope thereby conferring proper shape and mechanical rigidity to the nucleus. Lamin proteins are also shown to be related diversely to basic nuclear processes. More than 400 mutations in human lamin A protein alone have been reported to produce at least 11 different disease conditions jointly termed as laminopathies. These mutations in lamin A are scattered throughout its helical rod domain, as well as the C-terminal domain containing the conserved Ig-fold region. The commonality of phenotypes in all these diseases is characterized by misshapen nuclei of the affected tissues which might stem from altered rigidity of the supporting lamina hence lamins. Here we have focused on autosomal dominant Emery-Dreifuss Muscular Dystrophy, one such laminopathy where R453W is the causative mutation located in the Ig domain of lamin A. We have investigated by single-molecule force spectroscopy how a stretching mechanical perturbation senses the destabilizing effect of the mutation in the lamin A Ig domain and compared the mechanoelastic properties of the mutant R453W with that of the wild-type in conjunction with steered molecular dynamics. Furthermore, we have shown the interaction of Ig domain with emerin, another key player and interacting partner in the pathogenesis of EDMD, is disrupted in the R453W mutant. This altered mechanoresistance of Ig domain itself and consequent uncoupling of lamin A-emerin interaction might underlie the altered mechanotransduction properties of EDMD affected nuclei.